http://biscweb.uark.edu/ipinto/biol5334/5.pdf

Protein-protein interactions: methods for detection and analysis.

https://kirschner.med.harvard.edu/files/bionumbers/AlbeKRenzymeconc1989.pdf

Cellular concentrations of enzymes and their substrates

The mammalian pyruvate dehydrogenase complex (PDC) plays central and strategic roles in the control of the use of glucose-linked substrates as sources of oxidative energy or as precursors in the biosynthesis of fatty acids The activity of this mitochondrial complex is regulated by the continuous operation of competing pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP) reactions The resulting interconversion cycle determines the fraction of active (nonphosphorylated) pyruvate dehydrogenase (E1) component Tissue-specific and metabolic state-specific control is achieved by the selective expression and distinct regulatory properties of at least four PDK isozymes and two PDP isozymes The PDK isoforms are members of a family of serine kinases that are not structurally related to cytoplasmic Ser/Thr/Tyr kinases The catalytic subunits of the PDP isoforms are Mg2+-dependent members of the phosphatase 2C family that has binuclear metal-binding sites within the active site The dihydrolipoyl acetyltransferase (E2) and the dihydrolipoyl dehydrogenase-binding protein (E3BP) are multidomain proteins that form the oligomeric core of the complex One or more of their three lipoyl domains (two in E2) selectively bind each PDK and PDP1 These adaptive interactions predominantly influence the catalytic efficiencies and effector control of these regulatory enzymes When fatty acids are the preferred source of acetyl-CoA and NADH, feedback inactivation of PDC is accomplished by the activity of certain kinase isoforms being stimulated upon preferentially binding a lipoyl domain containing a reductively acetylated lipoyl group PDC activity is increased in Ca2+-sensitive tissues by elevating PDP1 activity via the Ca2+-dependent binding of PDP1 to a lipoyl domain of E2 During starvation, the irrecoverable loss of glucose carbons is restricted by minimizing PDC activity due to high kinase activity that results from the overexpression of specific kinase isoforms Overexpression of the same PDK isoforms deleteriously hinders glucose consumption in unregulated diabetes

Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms.

One of the accepted characterizations of the living state is that it is complex to an extraordinary degree. Since our current understanding of the living condition is minimal and fragmentary, it is not surprising that our first descriptions are simplistic. However, in certain areas of metabolism, especially those that have been amenable to experimentation for the longest period of time, the simplistic explanations have been the most difficult to revise. For example, current texts of general biochemistry still view metabolism as occurring by a series of independent enzymes dispersed in a uniform aqueous environment. This notion has been shown to be deeply flawed by both experimental and theoretical considerations. Thus, there is ample evidence that, in many metabolic pathways, specific interactions between sequential enzymes occur as static and/or dynamic complexes. In addition, reversible interactions of enzymes with structural proteins and membranes is a common occurrence. The interactions of enzymes give rise to a higher level of complexity that must be accounted for when one wishes to understand the regulation of metabolism. One of the phenomena that occurs because of sequential enzyme interactions is the process of channeling. This article discusses enzyme interactions and channeling and summarizes experimental and theoretical results from a few well-studied examples.

Macromolecular Compartmentation and Channeling

Complex I binds several mitochondrial NAD-coupled dehydrogenases.

Association between the α-ketoglutarate dehydrogenase complex and succinate thiokinase

A study on the physical interaction between the pyruvate dehydrogenase complex and citrate synthase

Interaction between the pyruvate dehydrogenase complex and citrate synthase

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1983.tb07748.x

István Alkonyi

Papers

Association between the α-ketoglutarate dehydrogenase complex and succinate thiokinase

A study on the physical interaction between the pyruvate dehydrogenase complex and citrate synthase

Interaction between the pyruvate dehydrogenase complex and citrate synthase

Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex.

Paracatalytic inactivation of pig heart pyruvate dehydrogenase complex.